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Comparison of the Effects on Mrna and Mirna Stability Arian Aryani and Bernd Denecke*

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Comparison of the Effects on Mrna and Mirna Stability Arian Aryani and Bernd Denecke* Aryani and Denecke BMC Research Notes (2015) 8:164 DOI 10.1186/s13104-015-1114-z RESEARCH ARTICLE Open Access In vitro application of ribonucleases: comparison of the effects on mRNA and miRNA stability Arian Aryani and Bernd Denecke* Abstract Background: MicroRNA has become important in a wide range of research interests. Due to the increasing number of known microRNAs, these molecules are likely to be increasingly seen as a new class of biomarkers. This is driven by the fact that microRNAs are relatively stable when circulating in the plasma. Despite extensive analysis of mechanisms involved in microRNA processing, relatively little is known about the in vitro decay of microRNAs under defined conditions or about the relative stabilities of mRNAs and microRNAs. Methods: In this in vitro study, equal amounts of total RNA of identical RNA pools were treated with different ribonucleases under defined conditions. Degradation of total RNA was assessed using microfluidic analysis mainly based on ribosomal RNA. To evaluate the influence of the specific RNases on the different classes of RNA (ribosomal RNA, mRNA, miRNA) ribosomal RNA as well as a pattern of specific mRNAs and miRNAs was quantified using RT-qPCR assays. By comparison to the untreated control sample the ribonuclease-specific degradation grade depending on the RNA class was determined. Results: In the present in vitro study we have investigated the stabilities of mRNA and microRNA with respect to the influence of ribonucleases used in laboratory practice. Total RNA was treated with specific ribonucleases and the decay of different kinds of RNA was analysed by RT-qPCR and miniaturized gel electrophoresis. In addition, we have examined whether the integrity observed for ribosomal RNA is applicable to microRNA and mRNA. Depending on the kind of ribonuclease used, our results demonstrated a higher stability of microRNA relative to mRNA and a limitation of the relevance of ribosomal RNA integrity to the integrity of other RNA groups. Conclusion: Our results suggest that the degradation status of ribosomal RNA is not always applicable to mRNA and microRNA. In fact, the stabilities of these RNA classes to exposure to ribonucleases are independent from each other, with microRNA being more stable than mRNA. The relative stability of microRNAs supports their potential and further development as biomarkers in a range of applications. Keywords: mRNA, miRNA, Ribosomal RNA, Ribonuclease, RNA stability, RNA integrity Background of their specific target genes [6]. The regulatory function of MicroRNAs (miRNAs) are small, non-coding RNAs which miRNA is differentiated into two distinct mechanisms: the are encoded within the genome. The primary transcript, inhibition of target gene expression (i) by inducing the deg- pri-miRNA, contains one or more hairpin structures and is radation of the target mRNA [4] and/or (ii) by inhibition cleaved to pre-miRNA [1,2]. Upon export to the cytoplasm, of target gene translation [7]. Partial complementary base dicer cleaves the pre-miRNA to mature miRNA, which pairing causes translational inhibition whereas perfect base usually has a length of about 22 ribonucleotides [1,3,4]. pairing causes degradation of the complementary target Previous studies have suggested that mRNA stability is mRNAs. It has long been assumed that translational inhib- an important control point for gene expression regulation ition is the dominant mechanism, however, recent studies [5]. The mature miRNA plays a key role in the regulation have shown that the degradation of target mRNA plays the major role in the inhibition of protein expression [8]. A widespread interest has developed in small non-coding * Correspondence: [email protected] Interdisciplinary Center for Clinical Research Aachen (IZKF Aachen), RWTH RNAs for cancer research and drug development. In Aachen University, Pauwelsstrasse 30, Aachen, Germany © 2015 Aryani and Denecke; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Aryani and Denecke BMC Research Notes (2015) 8:164 Page 2 of 9 particular, miRNAs appear to play a critical role in tumori- constituent of total RNA in mammalian cells [15]. For this genesis and are thus promising candidates as potential reason the quality of mRNA is generally extrapolated on therapeutic targets or as biomarkers [9,10]. Moreover, miR- the basis of the quality of ribosomal RNA, which can be NAs are able to regulate the expression of multiple genes analysed, for example, with microfluidic analysis (Agilent and hence affect disease pathways at multiple points [10]. 2100 Bioanalyzer). Therefore, miRNAs are promising tools in diagnostic and Representative electropherograms of our samples that therapeutic applications in a wide range of specializations, were analysed with the RNA6000 Nano labchip are including cancer research [11], cardiovascular disease [12], illustrated in Figure 1A. The electropherogram of the un- organ transplantation [13] and rheumatoid arthritis [14]. treated sample consists of four peaks: i) the loading Although miRNAs comprise an important class mole- marker at 25 nt, ii) the spiked tRNA at about 80 nt, iii) the cules used to uncover regulatory mechanisms in the cell, 18S ribosomal RNA and, iv) the 28S ribosomal RNA. The nearly nothing is known about their decay in comparison tRNA was added after the treatments in order to facilitate to mRNA under defined in vitro conditions. For this rea- the precipitation of RNA. It was clear that the 18S and son, we decided to enhance general knowledge about the 28S ribosomal RNAs were missing after NaOH treatment. stability of mRNA and miRNA in response to ribonucle- This was expected because NaOH, at the concentration ases commonly used in routine laboratory practice. We used, induces the hydrolysis of RNA in a manner that is were interested in finding out how a hypothetical contam- independent of size and sequence. Similar to NaOH, ination with ribonuclease might affect mRNA and miRNA RNase A-treated samples lacked both 18S and 28S peaks. stability. Furthermore, we investigated possible differences This indicated that both NaOH and RNase A treatments in the stability of different classes of RNA (i.e. miRNA, significantly affected ribosomal RNA. mRNA or ribosomal RNA). For this purpose, total RNA In the case of treatment with benzonase and RNase If, was treated in vitro with a range of ribonucleases under even though the ribosomal peaks were not detectable by identical experimental conditions and their effects were this method, the measured absorption was above the analysed by RT-qPCR. base line. This is caused by residues of ribosomal RNA. No specific changes in comparison to the untreated Results sample were observed in the electropherograms of the The integrity of ribosomal RNA is affected by treatment samples after treatment with RNase H, Exonuclease T or with specific ribonucleases Exonuclease T7. In contrast to mRNA, which represents only 0.5 to 3% of In order to determine the decay of ribosomal RNA the transcriptome, ribosomal RNA is the most prevalent after the treatments with a higher sensitivity, a relative Figure 1 The Stability of ribosomal RNA is affected in a treatment-dependent manner. (A) Representative electropherograms of untreated RNA (black) and RNA after treatment with either Benzonase (dark orange), NaOH (pink), RNase A (red), RNase H (grey), RNase If (bright orange), Exonuclease T7 (dark blue) or Exonuclease T (bright blue). Electropherogram of RNA ladder, used for size calculation indicated in the abscissa as kilobases (kb), is illustrated in green. Arbitrary units for fluorescence are given in the ordinate. The untreated control sample represents four peaks: the loading marker at 25 nt, the spiked tRNA at about 80 nt, the 18S and the 28S ribosomal RNA. (B) Detection of 18S ribosomal RNA using RT-qPCR. Percentage of intact 18S ribosomal RNA after indicated treatments is illustrated for three independent experiments with mean and SD (in red). The relative percentage of intact RNA in comparison to the untreated RNA is given in the ordinate. The green dotted line represents the 100% intact 18S ribosomal RNA obtained from the untreated RNA. Significances were calculatedbythetwotailedunpairedStudent’s t-test: * = p < 0.05; *** = p < 0.001. ExoT7 = Exonuclease T7;ExoT=ExonucleaseT. Aryani and Denecke BMC Research Notes (2015) 8:164 Page 3 of 9 measurement of 18S RNA was performed with RT- Nppb (Cq = 19.8 ± 0.8 cycles), Hprt (Cq = 24.7 ± 0.4 cy- qPCR (Figure 1B). The percentage of undigested 18S cles) and Polr2a (Cq = 26.9 ± 0.5 cycles). ribosomal RNA normalised to the untreated control is On the basis of our previous quantitative studies [19], presented in Figure 1B. It was observed that, similar to miR-1 and miR-208 (cardiac-specific expressed miRNAs) the results obtained by electrophoresis after treatment [20], sno202 (small nuclear RNA 202, often used as a with RNase A or NaOH, a significant reduction of ribo- normalization control) [21], and miR-501 [22] were in- somal RNA was detectable (a reduction of 77% and 66%, vestigated as indicators of miRNA stability. Figure 2B il- respectively). The observed difference in reduction be- lustrates the Cq values of the miRNA targets in the tween the two methods was probably caused by the untreated controls. Sno202 was more highly expressed higher sensitivity of the RT-qPCR. in heart tissue than the other miRNAs (Cq = 22.9 ± A similar effect could be observed after treatment with 0.5 cycles), followed by miR-1 (Cq = 23.3 ± 0.3 cycles) benzonase and RNase If. In contrast to a dramatic re- and miR-208 (Cq = 25.8 ± 0.2 cycles).
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